Major depressive disorder (MDD) is one of the most prevalent and debilitating illnesses world wide, affecting ~17 percent of the population and causing enormous personal and economic burden. The impact of MDD is underscored by the limitations of currently available medications, including low response rates, treatment resistance, and therapeutic time-lag (weeks to months). These data highlight a major unmet need for more efficacious and faster-acting antidepressants. Recent studies demonstrate that a single low dose of scopolamine, a muscarinic receptor antagonist, produces rapid and long-lasting antidepressant actions in treatment resistant patients. This rapid action, by a mechanism completely different from typical monoamine reuptake inhibitors, represents one of the most significant findings in the field of depression over the past 60 years. The mechanisms underlying the rapid antidepressant actions of scopolamine have not been identified, and the current application addresses this issue. Preliminary studies have found that scopolamine rapidly stimulates the mammalian target of rapamycin complex 1 (mTORC1), a pathway involved in synaptic protein synthesis, rapidly increases spine number and function of PFC neurons, and produces rapid antidepressant behavioral responses in rodent models. Moreover, the actions of scopolamine are blocked by rapamycin, demonstrating a requirement for mTORC1. Based on these findings, we hypothesize that the rapid antidepressant actions of scopolamine result from stimulation of mTORC1 and increased synaptic connectivity in PFC that occur via a burst of glutamate and release of brain derived neurotrophic factor (BDNF). This application describes an integrated multidisciplinary approach, including molecular, biochemical, electrophysiological, morphological, optogenetic, and behavioral studies to test this hypothesis. Aim 1 will characterize the time course and regional localization of scopolamine-stimulation of mTORC1, spine density, and behavioral responses, and confirm the requirement for mTORC1 and BDNF using a combination of pharmacological, viral vector, and mutant mouse approaches. The hypothesis that scopolamine can rapidly reverse the synaptic and behavioral deficits caused by chronic stress will also be tested. Aim 2 will determine the role of glutamate-AMPA receptor transmission, which has been implicated in activity-dependent stimulation of BDNF-mTORC1 signaling and synapse formation, using microdialysis and selective inhibitors. Microinfusions and optogenetic approaches will be used to test the role of glutamate transmission in subregions of PFC and to identify target circuits that underlie rapid antidepressant responses. Aim 3 will test if the glutamate burst occurs via muscarinic-1 (M1) receptors located on GABA interneurons, resulting in disinhibition of glutamate transmission, using cell specific virally expressed floxed shRNA, M1 deletion mutants, and cell specific Cre mice. Characterization of the signaling pathways for scopolamine will identify fundamental mechanisms for rapid acting antidepressants and novel targets for safer, rapid-acting agents.